Antibiotic and production thereof



Oct. 9, 1962 A. J. sHAY ETAL ANTIBIOTIC AND PRODUCTION THEREOF FiledMarch 14, 1960 OWN DOQ OOM. OOO\ 00mm nos* 060m. 009m.

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United States Patent O 3,057,779 ANTIBIOTIC AND PRODUCTION THEREOFAnthony Joseph Shay, Pearl River, James Alfred Lowery,

New City, Nestor Bohonos, Nanuet, and Edward James Backus, Pearl River,N.Y., assgnors to American Cyanamid Company, New York, N.Y., acorporation of Maine Filed Mar. 14, 1960, Ser. No. 14,990 7 Claims. (Cl.167-65) This invention relates to a new antibiotic and to its productionby fermentation, to methods for its recovery and 4concentration fromcrude solutions, to processes for its purication and to methods for thepreparation of its salts.

The present invention includes within its scope the antibiotic in diluteforms, as crude concentrates, and in pure crystalline forms. These novelproducts are active against a variety of microorganisms includinggrampositive bacteria. The eiects of the new antibiotic on specicmicroorganisms together with the chemical and physical properties of theantibiotic diiferentiate it from previously described antibiotics.

The new antibiotic called aspartocin (formerly designated A8999) isformed `during the cultivation under controlled conditions by the1microorganisms Streptomyces griseus var. Spiralis and Streptomycesviolaceus var. aspartocz'ncus. The following is a general description ofthe organism Streptomyces grseus var. Spiralis based on Mice thediagnostic characteristics observed. The underscored descriptive colorsare those of Ridgway, Color Standards and Color Nomenclature.

Amount of growth: Growth moderate to good on many media, spreading onstarch-containing media; poorer, restricted growth on certain syntheticmedia.

Aerial mycelium and/or spore color: Spores en masse are Pale Olive-Buti;non-sporing aerial mycelium colorfless to whitish.

Soluble pigments: None.

Reverse color: In shades of bui to yellowish on most media.

Miscellaneous physiological reactions: No growth on cel lulose; completeliquefaction of gelatin; formation of acid curd and clearing of purplemilk; and negative HZS reaction on peptone-iron agar media.

Morphology: Sporiferous appendages arise from aerial mycelium as coilsof a few turns or short spirals. Spores elongate, rod-like, truncate(1.0-1.2;L 0.6p), spaced regularly apart in chains, smooth Walled.

Temperature eifect: Optimal range for growth and sporulation 18 C. to 37C. Maximum temperature for growth 42 C., no growth at 48 C. Minimumtemperature 10 C., no growth at 4 C.

The cultural characteristics of the new variety of S. grz'seus are setforth in the following table. The underscored descriptive colors weretaken from Ridgway.

TABLE l Streptomyces Griseus var. Spiralis Medium Amount oi' growthAerial mycelium and spore color Solublet Reverse color Remarks pigmenWaksmans starch agar Good; spreading Pale Olive-Buti; sporulation heavy.None.... Deep Colonial Moderate starch hydrolysis.

Buff

Asparagine dextrose meatextract Moderate Pale Olive-Buff; sporulationmoderate. do Colonial Bumu" Limited colorless agar.

exudate.

Acid asparagine dextrose meat Moderate restricted... Pale Olive-Buff;sporulation very do do extract agar.

slight.

Synthetic agar (Czapeks agar) Thin; restricted White powdery aerialmycelium; do. White feather-y margins.

Emerson's agar Moderate M le Olive-Buff; sporulation light; do ColonialBut colonies lightly rimose.

Nutrient agar Poor, thin growth.--.. None do.. Colorless Calcium malateagar Moderate; restricted... Pale OliveBui; sporulation light--.Moderate /zone oi'. malate clearing.

Cobalt amidex agar Good; spreading Olive-Buti to PaleOlive-Bu;spo1-ulado. Im XuO-W Lightly zonte.

tion heavy. Potato dextrose agar Moderate Pale Olive-Buti; sporulationmoderate. do.. C Ol-- g2g-"'- Bennetts agar .do Pale OlivcBufi;sporulation heavy. do. m m Lieniltegtglorless Corn steep liquor agar doPale Olive-Buff: sporulation moderato. -do glei-nl 921i Sabouraudsmaltose do Aerial mycelium white; no sporulado. Mm'

tion; central colony zones barren.

Yeast-malt agar do Pale Olive-Buti;sporulationheavy ...do do Moderatecolorless exudate.

In gross appearances this new strain bears close resemblance to severalstrains of S. grseus. Spore coloration, reverse colors and growth habitsare similar. However, when the sporophores are compared microscopically,all

media; spreading on Waltsmans Starch, Czapeks, Corn Steep Liquor, andCobalt-amidex agars.

Aerial mycelium and/ or spore color: Spores en masse Light Mouse Gray onmost media which support sporuof the S. griseus strains have straight toilexuous chains of 5 lation.

globoSe to eliPical Spores, in contrast lo the coiled and Solublepigment: In reddish to vinaceous to bluish shades spiralled chains oftruncate, rod-like spores of the new on media which permit pigmentformatiOn.

Stran- The Combination of l1elicoiderl SPoroPlloreS and Reverse color:In reddish to vinaceous to bluish or even bacillary-type sporesjustifies varietal status for this strain brownish shades, dependingupon the medium.

in the S- gfSellS conlPleX- The narne S- gr'l'seus Ver 10 Miscellaneousphysiological reactions: Moderate starch spirals has been chosen to bedescriptive of the sporiferhydrolysis; no H25 produced; moderato gelatinliquoous structures of the organism. The new isolate, when faction;oelluloso decomposed,

keyed according to WnkSrnan and Lncllevalier, Aciino' Morphology:Sporiferous appendanges arising as coils mycets and Their Antibiotics,falls into subdivision III- or loose spirals from aeria1 hyphae. Sporessmooth,

no Soluble Pigment in organic media-and iiS best into 15 typicallyglobose, but with occasional elliptical ones the category (g)growthcolorless to yellowish to olivein Chain, 1.0-12@ buff-in Wbcb S- grfseuriS lille representative SPecieS- Temperature relations: Growth fair at24-28" C.; good The following is a general description of the organismat 32 37 C Streptomyces violaceus var. aspartocinicus based on the TheCultural, physiological and morphological characdiagnosticcbaracleIiSicS observed The underscore de- 20 teristics of the organismStreptomyces volaceus var.

scriptive colors were taken from Ridgway. aspartocz'nicus are set forthin the following tables. The

Amount of growth: Moderate to good growth on most underscoreddescriptive colors were taken from Ridgway.

TABLE 2 in Petri Dis/zes Medium Amount of growth Aerial myeelium andspore color Soluble pigment Reverse color Remarks Waksmans starch agarGood; spreading Spores Light Mouse Gray; dark None Dusky Drab ModerateStarch broadly. non S hydrolysis.

poring sectors prominent, sporulation moderate Asparaglne dextrose meatModerate Spores Light Mouse Grey; spor` do Light brownish Faintlyzonate; extract agar. mation-n thinwith Vinaceousmargins thin,

Gray patches. submerged' Acid asparagine dextrose Poor; restricted Nonedo Colorless meat extract agar.

Synthetic agar (Czapeks Good; spreading Spores Light Mouse Gray; mod-Blush-purple; Anthracene purple 'man' crate sporulation withsectormoderate' ing.

Emerson's agar Good Spores Light Mouse Gray; spor- Reddish; very DeenMouse Gray.

uletion heavy. hght Nutrient agar Moderate Trace of whitish to grayishaerial None COlOrleSS t0 growth. brownish. Calcium malate -do Very poorsporulation; aerial -do VDBCCOnS-Fnwn" Large zone oi growth Pale MouseGray. malate clearing. Yeast extract-malt extract .do Spores Light MouseGray; spordo .r F 11500115 t0 Bone Thin submerged agar' ulatioumoderate. @Pi mnrgms. Waksmans glucose agar -do Spores Pale Mouse Gray;spor. Vinaceous mod. Deep Brownish Margnilrzl onesd 's erate. wr e anulatlon hebt Db.: without aerial growth. Kralnskys dextrose agar doSpores Pale Mouse Gray; spor- None plnkish Buff to ulation very light.Fna-wn c ol-oz". Potato dextrose agar do Aerial growth seanty; sporula-Pinkish-vinaceous Vimoeouoslote Thin submerged t1on Light Mouse Gray,light margins s arse-' to Deep Slnty p Brown.

Bennetts agar Good Spores Light Mouse Gray; spor- None DospMouso Gi-syDo.

ulation moderate.

Corn steep liquor agar-...-..- Moderate; thin sporulation Light MouseGray do Fawn color i D0 spreading to Light Drab; sporulation moderate.

Sabouraudsmaltose agen... Good Spores Light Mouse Grey; spordo mm1-uiaton moderate.

Cobalt-amidex agar Moderate; spreading.- Light Mouse Gray Reddish; lightVinaousslnte Margine thin,

to Deep Slaty submerged. Brown.

Czapeks-Dox Mannitol agar. Good; spreadlng- Aerial myeelium white withVineceous; light Vinaeeous-Slate.-.

spores Pale Mouse Gray; sporulation moderate.

TABLE 3 Observations of Some Miscellaneous Physiological Tests onStreptomyces Violaceus Var. Aspartocinicus Medium Amount of growthAerial mycelium and spore color Soluble pigment Revlerse Remarks co orPeptonedron agarm. Good Negative HgS reaction. Gelatin Moderate NoneNone Moderateliqueactlon; part of ltube not liquied.

Potato plugs Good; covering entire exposed Light Mouse GrayinsporulatingReddish: light.-. Plug Slightly darkened.

surface. a,

Carrot plugs Moderate; covering entire .do None exposed surface.

Litmus milk Moderate Clearing of purple milk, and

curd precipitated; pH 7.0.

Cellulose,1 (filter do None Vinaceous;light Filter paper decomposed inpaper in Czapeks growth areas. solution).

l Incubation 21 days.

TABLE 4 Morphological Features of Streptomyces Violaceus Var.Aspartocinicus [Medium: Waksmans Starch agar] Culture No. Aerialmycelium Spore shape Spore Remarks size, ,u

Streptomz/ces violaccus Sporifcrous appendages arising as Typicallyglobose, but with occa- 1.0-1.2 spores, when viewed under the coils orloose spirals from aerial var. aspartocnicus hyphae.

sional elliptical spore in chains.

electron microscope, had smooth Walls.

Viable cultures of Streptomyces grseus var. sprals and Streptomyceslvz'olaceus var. aspartocnicus have been deposited with the AmericanType Culture Collection in Washington, D.C., where they have beenassigned ATCC accession numbers 13733 and 13734, respectively.

It is to be understood that for the production of the antibiotic of thisinvention, the present invention is not limited to the above organismsor to organisms fully answering the above growth and microscopiccharacteristics which are `given for illustrative purposes. In fact, itis desired and intended to include the use of mutants produced from thedescribed organisms by various means, such as X-radiation, ultravioletradiation, nitrogen mustard, phage exposure and the like.

The cultivation of the organisms S. giseus var. spiralis and S. volaceusvar. aspartocnicus may take place in a variety of liquid culture media.Media which are useful for the production of the novel antibioticinclude an assimilable source of carbon such as starch, sugar, molasses,glycerol, etc., an assimilable source of nitrogen such as protein,protein hydrolysate, polypepetides, amino acids, corn steep liquor,etc., and inorganic anions and cations, such as potassium, sodium,calcium, sulfate, phosphate, chloride, etc. Trace elements such asboron, molybdenum, copper, etc., are supplied as needed in the form ofimpurities by other constituents of the media. Aeration in tanks andbottles is provided by forcing sterile air through or onto the surfaceof the fermenting medium. Further agitation is provided in tanks by amechanical impeller. An antifoaming agent such as 1% octadecanol in-lard oil may be added as needed.

IFor the shaker flask fermentations, 100 milliliter portions of thefollowing liquid inoculum in 500 milliliter asks are inoculated with anagar slant of the culture.

Liquid inoculum: Grams per liter 'Calcium carbonate 3 The flasks areincubated on a reciprocating shaker and agitated vigorously for 48hours.

For the production of the antibiotic in tank fermenters, the followingfermentation medium is preferably used.

Fermentation medium: Grams per liter Calcium carbonate 1 Each tank isinoculated with 1% of a culture broth fermented as described above forshaker ask fermentation. Aeration is supplied at the rate of 0.2-2.0volumes of sterile air per volume yof broth per minute and the broth isagitated by an impeller driven at about -160 r.p.m. The temperature ismaintained at 2035 C., usually at 28 C. The fermentation may becontinued for from 24-240 hours at which time the activity is harvested.

After the fermentation is completed, the culture broth containing theantibiotic of this invention is preferably Ifiltered at pH 5.0 to removethe mycelium from the broth. Diatomaceous earth or any of theconventional filtration aids may be used to assist the filtration whichis carried out using standard equipment. Thereafter, the antibiotic mayIbe recovered from the mycelial cake by appropriate extractionprocedures.

The antibiotic of this invention may be extracted from the filter cakewith water at pH 1 2 or 9-10` and back extracted into an immisciblesolvent such as butanol at pH l-3. Roughly, 80-9i)% `of the activity iscontained in the filter cake.

When the butanol solution is concentrated `(1/25 to 1%@ its volume) toanhydrous butanol, the l,antibiotic is precipitated. Thebutanol-precipitate mixture is stirred with acetone or petroleum etherand the precipitate removed by centrifugation. The antibiotic is thenWashed with acetone, and dried yielding an amorphous product.

Alternatively, calcium chloride or other salts may be added at thisstage of the operation or earlier if desired to facilitate precipitationand/ or crystallization.

Purification of the antibiotic may be obtained by crystallization of thecalcium salt from solvent mixtures such as butanol-water ormethanol-Water in a standard manner.

The novel antibiotic of this invention is composed of the elementscarbon, hydrogen, nitrogen, sulphur and oxygen. Elemental analyses showvalues in substantially the following proportions by weight:

The product has an optical rotation of [a]D25=-{26.4 (6., 2.1% solutionin methanol). The product shows no characteristic ultravioletabsorption. The product is soluble in water below pH 3.0 and above pH3.6. The isoelectric point is near 3.3. The free acid is soluble inmethanol, ethanol, butanol, glacial acetic acid, water and wet butanol.It is soluble to the extent of 0.6 mg./ml. in acetone and 0.1 mg./ml. inethyl acetate and ether.

An infrared absorption spectrum of the free acid was prepared in astandard manner by mixing with crystals of KBr and pressing into a disc.The compound exhibits characteristic absorption in the infrared regionof the spectrum at the following wavelengths expressed in microns: 3.07,3.45, 6.03, 6.53, 6.89, 7.15, 8.10 and 9.82. The infrared curve is shownin the accompanying drawmg.

Microbiological and paper chromatographic analyses of acid hydrolysatesindicate the following amino acid contents:

Three other ninhydrin positive components have been identified asD-a-pipecolic acid, a[L]-methylasparatic acid and a-diaminobutyric acid.

The antibiotic shows the following R1 values in the solvent systemsindicated below:

Solvent system s-Collidine saturated with water.

0.2 M phosphate pH 6.0.

100 n-amyl acetate.

30 dibutyl ether.

acetic acid.

50 water.

100 chloroform.

35 90% phenol.

4 acetic acid.

2 pyridine.

50 water.

200 benzene.

100 cyclohexane.

acetic acid.

50 water.

200 rncresol.

1 HOAc.

1 pyridine.

100 water.

200 nabutanol.

50 pyridine.

200 water.

200 m-cresol.

100 0.1 M phosphate pH 7.0. v5% NaCl.

200 m-cresol.

100 0.1 M phosphate pH 6.0.

Solvent system 200 n-butanol.

50 pyridine.

50 acetic acid.

100 water.

Water adjusted to pH 2.3 with tartaric acid.

200 n-butanol.

100% NaHCO3.

100 l,2dchloroethane.

100 carbon tetrachloride.

25 acetic acid.

50 water.

100 CHCI3.

40 pyridine.

40 HOAc.

v 50 water 0.97 200 phenol.

50 m-cresol.

8 HOAc.

8 pyridine.

50 water.

The novel antibiotic is clearly distinguished from other antibiotics byits chemical and physical properties described above, by itsantimicrobial spectrum and by paper chromatography.

Although aspartocin has chemical and biological properties similar toamphomycin, the two antibiotics can be readily separated by paperchromatography or paper electrophoresis. Individual and mixed spots ofaspartocin and amphomycin are subjected to descending paperchromatography with 5 percent NH4Cl solution as development of the paperchromatogram is terminated after 6-7 Rf Value r hours. This isapproximately double the time necessary for the solvent to descend tothe bottom of the paper strip. Bioautography indicates that aspartocinmoves approximately one inch, whereas amphomycin travels six to seveninches. Zone electrophoresis on paper wetted with 5 percent acetic acidshows that amphomycin has approximately six times the mobility ofaspartocin towards the cathode.

The antibacterial spectrum of the antibiotic of this invention wasdetermined by the serial broth dilution assay technique and is presentedin the table below which shows the minimal inhibitory concentrationrequired to inhibit the growth of representative microorganisms intrypticase soy broth:

TABLE 5 Minimal inhibitory Organism: concentration, mcg/ml.Staphylococcus aureus ATCC 65 -38 15.5 Staphylococcus aureus 2091J 15.5Streptococcus pyogenes C-203 2.0 Streptococcus pyogenes NY-S 4.0Corynebacteriumy xerosis NRRL B-1397 1.0 Bacillus cereus 4.0 Sarcinaluzea 2.0 Bacillus polymyxa 4.0 Bacillus megatherium 0.25 Erysipelothrixrhusophathiae 0.5 Bacillus subtilis ATCC 6633 4.0 Klebsiella pneumoniae250 Pastearella multocida 250 Salmonella gallinarum 250 Escherichia coli250 Proteus vulgaris 250 Candida albicans 250 Mycobacterium ranae 62.0Mycobacterium 607 62.0

The new antibiotic is highly active in vitro against Staphylococcitested including S. aureus and S. albus, coagulase positives andnegatives. Streptococci including nt-hemolytic, -hemolytic andnon-hemolytic strains are sensitive to the new antibiotic. Many of theabove-mentioned organisms are isolates obtained from clinical sourcesand are to some extent resistant to either penicillin or thetetracycline antibiotics. The antibiotic is also highly activeintraperitoneally and subcutaneously against three standardizedinfections in mice, Streptococcus C-203, Diplococcus pneumoniae SVl andStaphylococcus aureus.

Aspartocin has also been found to be substantive to cloth such as cottonfabric and may be added to cotton cloth for the purpose of rendering thecloth bacteriostatic in substantially the same manner as has been founduse ful with the'antibiotic neomycin.

'Ihe novel antibiotic of this invention is especially valuable becauseof its growth-promoting effects in fowl, i.e., chickens and in baby pigsand swine. The antibiotic may be added to a purified chick dietcontaining casein as the protein source and sucrose as the carbohydratesource supplied with all of the known vitamins and minerals. lt has beenfound that the antibiotic may be usefully added to such diets in amountsas little as I or l0 parts per million per kilogram of diet. More may beadded if desired but so far no need has been found for adding more than500 milligrams per kilogram of diet. The percent increase in weight ofthe chickens over the controls ranges from about 25% to 30%. Theantibiotic has been found to produce significant increases in rate ofgain in baby pigs when added in the ratio of l0-30 grams per ton of feedcomparable to that of the effects obtained with chlortetracycline.

Aspartocin has not as yet been demonstrated to be useful in humantherapy.

The invention will be described in greater detail in conjunction withthe following specific examples.

EXAMPLE 1 Inoculum Preparation A typical medium used to grow the primaryinoculum is prepared according to the following formula:

Water to 1,000 mililiters.

A yeast-malt agar slant of a culture of the species S. griseus var.spiralis is incubated for a week. At this time the spores and myceliumare transferred to two 500 milliliter flasks which contain 100milliliters of the above medium. The flasks are placed on areciprocating shaker and agitated vigorously for 48 hours at 28 C. Theflask inocula are transferred to 9 liter bottles which contain 6 litersof the above liquid medium. These bottles are aerated for 24 hours toencourage further growth. At the end of this time the 9 liter bottlesare used to seed fermentor tanks.

EXAMPLE 2 Fermentation A fermentation medium is prepared according tothe following formula:

Grams Molasses 20 Corn starch 10 Bactopeptone l0 Calcium carbonate 1Water to 1,000 milliliters.

EXAMPLE 3 Isolation Two hundred liters of fermented mash are mixed with6000 grams of diatomaceous earth, adjusted to pH 5.0, filtered and thefiltrate discarded. The mash cake is washed twice-first with 40 litersof H2O at pH 5.0 and then with 40 liters of acetone. Both washes arediscarded. The mash cake is extracted twice with 5'0' liters of H2O atpH 2.0 adjusted with H2804, and once with 45 liters of H2O at pH 9.5adjusted with NaOH. The acid H2O and alkaline H2O extracts are eachextracted with 1/2 volume of n-butanol at pH 2-3. The separated butanolextracts are adjusted to pH 5 6 and concentrated to 1/25 to 1/50 oftheir volume causing the antibiotic to precipitate. The butanolconcentrate of the acid H2O extract is stirred with three volumes ofacetone, and the precipitate removed by centrifugation, washed withacetone and dried. The yield of product is 151 grams. The butanolconcentrate of the alkaline H2O is stirred with three volumes ofpetroleum ether, the precipitate centrifuged off, washed with acetoneand dried. The yield of product is 41 grams.

EXAMPLE 4 Isolation of the Calcium Salt Ten grams of calcium chlorideand 300 grams of diatomaceous earth are added to 10 liters of fermentedmash. The mixture is stirred, adjusted to pH 5.0 and maintained at thispH for 10 minutes during stirring. The precipitated `calcium salt isfiltered off with the mash cake and the spent filtrate discarded. Themash cake is washed by stirring in 2 liters of H2O at pH 5.0. Afterltration, the inactive H2O wash is discarded. The mash cake is furtherwashed by stirring with 2 liters of acetone. The mash cake is filteredand the inactive acetone wash discarded. The mash cake is extractedtwice by stirring l0 minutes with 2 liters of H2O at pH 1.0 adjustedwith HCl. The antibiotic enriched acid H2O is extracted with 1/2 volumeof nebutanol. The butanol extract is adjusted to pH 5 .0 andconcentrated under reduced pressure with the `addition of H2O tomaintain a wet butanol solution. The solution is concentrated toapproximately 500 milliliters and a wet butanolic solution of CaClZ isadded at pH 5.0 until there is no further precipitation. Theprecipitated calcium salt is removed by centrifugation, washed with wetbutanol, followed by an acetone wash and dried. The yield of crudecrystalline product is 6 grams. This product assays approximately pure.

EXAMPLE 5 Crystallzation 0f the Calcium Salt Forty grams of productprepared according to the procedure of Example 3, is dissolved in 200milliliters of H2O. An aqueous solution .of 40 grams of CaClz is addedand the solution adjusted to pH 9.0 with NaOH and filtered. The filteredsolution is readjusted to pH 5.0 with HCl and the precipitated calciumsalt is removed by centrifugation. The precepitate is dissolved in 400milliliters of H2O at pH 2.5 and 200 milliliters of methanol added. Thesolution is filtered and the methanol-H2O solution adjusted to pH 5.0and stored at 5 C. The crystalline calcium salt is removed bycentrifugation. The crystalline product is dissolved in 200 millilitersof methanol at pH 3.0 adjusted with HC1. The solution is stirred with 4grams of carbon and filtered. The filtered solution is adjusted to pH5.0 using NHZOH. An immediate crystalline precipitate -is formed whichis removed by ltration washed with methanol and dried. The first crop ofcrystalline calcium salt yields 3.85 grams. The mother liquor of thefirst crop of crystals is stored at 5 C. for 16 hours. A second crop ofcrystalline calJ cium salt is removed by filtration, washed withmethanol and acetone and dried. The second crop yields 3.37 grams.Additional crops are recovered by concentrating ll' the mother liquorunder reduced pressure and storing at 5 C.

EXAMPLE 6 Preparation f the Free Acid of Aspartocin One gram of thecalcium salt, similar to that prepared according to the procedure ofExample 5, is dissolved in 25 milliliters of H2O by adjusting with HC1to pH 1.5. The aqueous solution is adjusted to pH 3.0 and mixed with 15milliliters of an aqueous solution saturated with NaCl. The precipitatedfree acid is washed freely with H2O. The precipitate is dissolved in 50milliliters of methanol, filtered and concentrated to approximately 10milliliters. Forty milliliters of H2O is `added to the methanol solutionprecipitating the free acid. The precipitate is Washed with H2O,followed by an acetone wash, removed by centrifugation and dried. Yieldof the free acid is 340 milligrams. The chemical analysis of thisproduct and its other chemical, physical and biological properties havealready been described.

EXAMPLE 7 Preparation 0f the Pcrate Salt Ten grams of product preparedaccording to the procedure of Example 3 is dissolved in 250 millilitersof H2O at pH 2.0. An aqueous solution saturated with picric acid isadded until no further precipitate forms. The precipitate is removed bycentrifugation and washed with H2O. The picrate is dissolved in 200milliliters of acetone, concentrated under reduced pressure, to about-100 milliliters, and the picrate precipitated by the addition of H2Ountil precipitation is complete. The supernatant is decanted and `thepicrate dissolved in 200 milliliters of acetone. Butanol is added, andthe solution concentrated under reduced pressure to approximately 100milliliters of an anhydrous butanol solution. The butanol solution ismixed with three volumes of ether, the precipitated picrate removed bycentrifugation, washed with ether and dried. The yield of picrate is 8.5grams.

EXAMPLE 8 Preparation of the Sodium and Potassium Salts One hundredgrams of the calcium salt similar to that described in Example 4, isdissolved in 1 liter of H2O at pH 2.0 adjusted with HC1. The aqueoussolution is adjusted to pH 3.3 and extracted 2 times with approximately1 liter of butanol each time. The butanol extract after washing with H2Oat pH 3.4, is dried over NagSO.,l and ltered.

A 1A portion, 777 milliliters, of the butanol solution is stirred with200 milliliters of H2O, adjusted with NaOH to pH 8.5 and concentrated toapproximately 200 milliliters of an anhydrous butanol solution. Acrystalline sodium salt which precipitates is removed by centrifugation,washed with butanol, ethanol, acetone and dried. The yield ofcrystalline sodium salt, is 3.0 grams. A second crop of sodium salt iscollected by stirring the mother liquor with 10 volumes of acetone andremoving the precipitate by centrifugation. The precipitate is washedwith acetone and dried to yield 13.1 grams of material.

A potassium salt is prepared from a second 1/s portion of the butanolsolution by the same procedure except that KOH is used to adjust to pH8.5.

Five hundred milligrams of the free acid of aspartocin is dissolved in 5milliliters of ethanol. A saturated ethanolic solution of KOH is addeddropwise to the ethanol solution of aspartocin until no furtherprecipitate results. The precipitated potassium salt is removed bycentrifugation, washed with 8 milliliters of ethanol and 10 millilitersof acetone. The salt is dried under vacuum to yield 220 milligrams.

The sodium salt is prepared as described above using the sameconcentration of free acid and adding a saturated ethanolic solution ofNaOH to form the salt. The yield of dried sodium salt is 185 milligrams.

l 9 kd EXAMPLE 9 Production of Aspartocn by Streptomyces violaceus var.aspartocinicus The conditions of inoculum preparation and fermentationare the same as previously described in Examples 1 and 2 of thisapplication. The culture is fermented in two tanks using liters offermentation medium in each tank.

Caco3 3.o

After 137 hours of fermentation, the tank mashes are harvested, pooled,and processed as follows:

Isolation and Pm'ication of Aspartocin grams of calcium chloride and5700 grams of diatomaceous earth are added to 190 liters of fermentedmash. The mixture is adjusted to pH 5.5, stirred for 20 minutes andfiltered. The precipitated antibiotic is filtered oit with the mash cakeand the spent filtrate discarded. The mash cake is washed with 60 litersof water at pH 5.0-5.5 Iand iiltered. The inactive water wash isdiscarded. The mash cake is extracted twice by stirring 20 minutes with60 liters of water at pH 9.8-10.0. After filtration the alkaline waterextracts are pooled, adjusted to pH 1.0-3.0 with HC1 and extracted twicewith 1A volume of n-butanol. The 65 liters of pooled butanol extract are:adjusted to pH 5 .0-7.0 and concentrated under reduced pressure toapproximately 4 liters of anhydrous butanol. The antibiotic precipitatesand is removed by centrifugation and the butanol supernatant discarded.The precipitate is dissolved in approximately 500 milliliters of waterat pH 1.5 and the solution is filtered. The aqueous solution isextracted 3 times with 300 milliliter portions of n-butanol. The butanolextract 950 milliliters, is adjusted to pH 5.0 to 5.5 and stirred with50 milliliters of a wet butanol solution containing 3 grams of calciumchloride at a pH of 5.0-5.5. The precipitated calcium salt is removed bycentrifugation, washed with wet butanol, washed again with acetone anddried. The yield of product s 7.2 grams.

This application is a continuation-in-part of our copending applicationSerial No. 773,957, led November 14, 1958, now abandoned.

We claim:

1. A substance effective in inhibiting the growth of gram-positivebacteria having an isoelectric point of about pH 3.3, said substancebeing soluble in water, methanol, ethanol, butanol and glacial aceticacid, slightly soluble in acetone, ethyl acetate and ether, saidsubstance containing the elements carbon, hydrogen, nitrogen, sulphurand oxygen lin the following proportions by weight:

said substance having an optical rotation [a]D25=-|26.4 (c., 2.1%solution in methanol), said substance containing the followingcomponents: L-aspartic acid, L-proliue, L-valine, glycine, D-a-pipecolicacid, a[L]-methyl aspartic acid `and ,-diaminobutyric Iacid, theirstfour components being in the molar ratio of 4:1:1:2, respectively, andsaid substance when suspended in 'a potassium bromide pellet exhibitscharacteristic absorption in the infrared region of the spectrum yat thefollowing wavelengths expressed in microns: 3.07, 3.45, 6.03, 6.35,6.89, 7.15, 8.10 and 9.82, Iand which is produced by cultivating amicroorganism selected from the group consisting of Streptomyces griseusvar. sprals and Streptomyces violaceus var. aspartocinz'cus in anaqueous nutrient medium containing assimilable sources of carbohydratenitrogen, and inorganic salts under submerged aerobic conditions untilsubstantial antibacterial activity is imparted to said medium, and thenrecovering the so-produced substance from the medium.

2. A salt of said substance as defined in claim l.

3. A process for the production of aspartocin which comprisescultivating a microorganism selected from the group consisting ofStreptomyces griseus var. spiralz's and Streptomyces violaceus var.aspartocnz'cus in an aqueous nutrient medium containing :assimilablesources of carbohydrate, nitrogen and inorganic salts under submergedaerobic conditions until substantial antibacterial activity is impartedto said medium.

4. A process for the production of aspartocin which comprisescultivating Streptomyces violaceus var. aspartocincus in an aqueousnutrient medium containing 14 `assimilable sources of carbohydrate,nitrogen and inorganic salts under submerged aerobic conditions for aperiod of from 24 to 240 hours and Iat a temperature of from 20 to 35C., and recovering the antibiotic so-produced from the fermentationbroth.

5. A process for the production of aspartoclin which comprisescultivating Streptomyces griseus var. spirals in an yaqueous nutrientmedium containing assimilable sources of carbohydrate, nitrogen andinorganic salts under submerged aerobic conditions for a period of from24 to 240 hours and at `a temperature of from 20 to 35 C., andrecovering the antibiotic so-produced from the fermentation broth.

6. A process 'as in claim 5 in which the `antibiotic is recovered fromthe fermentation broth by ltening the broth, extracting the antibioticlactivity from the filter cake with Water, back-extracting theantibiotic activity into an immiscible solvent, and separating theantibiotic activity therefrom.

7. A process las in yclaim 6 in which a calcium salt is ladded to thefermentation broth so that aspartocin calcium salt is produced.

References Cited in the le of this patent Pridham et al.: AppliedMicrobiology, vol. 6, pages 52-79, 1958.

1. A SUBSTANCE EFFECTIVE IN INHIBITING THE GROWTH OF GRAM-POSITIVEBACTERIA HAVING AN ISOELECTRIC POINT OF ABOUT PH 3.3, SAID SUBSTANCEBEING SOLUBLE IN WATER, METHANOL, ETHANOL, BUTANOL AND GLACIAL ACETICACID, SLIGHTLY SOLUBLE IN ACETONE, ETHYL ACETATE AND ETHER, SAIDSUBSTANCE CONTAINING THE ELEMENTS CARBON, HYDROGEN, NITROGEN, SULPHURAND OXYGEN IN THE FOLLOWING PROPORTIONS BY WEIGHT: